BCNU, a widely-used antitumor drug, has previously been reported to cause irreversible inhibition of glutathione (GSSG) reductase in liver and red blood cells. GSSG reductase is responsible for maintaining the intracellular glutathione redox status and thereby has a major role in protecting the cell from oxidant damage. Because one of the most prominent clinical side-effects of BCNU is pulmonary toxicity, we have examined the effects of the drug on pulmonary GSSG reductase and GSH/GSSG ratio. A. In Vivo Studies: Single doses of 8 mg BCNU/kg inhibited lung GSSG reductase by 11% and 82%, respectively. The inhibition was very persistent, lasting up to 7 days after a single dose of BCNU. The effect of multiple doses of BCNU on GSSG reductase and GSH/GSSG levels was also studied. The multi-dosing regimen of 5 mg BCNU/kg/week for 6 weeks results in the development of severe pulmonary damage. After 3 weeks of BCNU dosing, GSSG reductase activity decreased to 50% of control values and GSSG levels rose to 227% of control values. After 6 weeks of BCNU, GSSG reductase activity decreased to 22% of control values. B. In Vitro Studies: BCNU also inhibited pulmonary GSSG reductase in vitro. The inhibition was dependent on length of incubation time and the concentration of BCNU. Other nitrosoureas, methyl-CCNU, also were able to inhibit GSSG reductase while streptozotocin and chlorozotocin had little, if any, effect on GSSG reductase activity. The in vitro inhibition can be mimicked by N-ethyl-maleimide which binds to free sulfhydryl groups. Addition of sulfhydryl donors, such as GSH and dithiothreitol, into the incubation media prevents the inhibition of GSSG reductase by BCNU. The glutathione redox system has been proposed to be important as an antioxidant defense mechanism in pulmonary cells and impairment of this system by BCNU might ultimately lead to or contribute to pulmonary toxicity.